The organization of cells into a multicellular form was a major event in evolution allowing both an increase in size of an organism and an increase in the number of specialized functions performed by an organism. The cellular slime molds occupy a unique position in evolution, since they normally proceed from a unicellular ameba to a multicellular form, called an aggregate, before differentiating into two specialized cell types. Thus they provide a manageable primitive system for studying how cells can achieve multicellularity. The primary trigger for aggregation in this system is starvation. However, most studies of aggregation in the slime molds have been concerned with the chemotactic mechanism responsible for collecting the cells into an aggregate. Very little is known about the events that occur after starvation and prior to chemotaxis. In Polysphondylium violaceum an aggregation stimulating factor (called D factor) has been described. This factor stimulates aggregation of an aggregateless mutant (aggA) and is produced during the preaggregation period prior to production of the chemoattractant. Preliminary physical and physiological experiments suggest that D factor may be a protohormone-like molecule involved in triggering aggregation. In specific, we will attempt to purify and identify the factor, understand the genetic complexity of its production, and study its relationship to other environmental conditions known to affect aggregation.